Liquid amnion transplant product

ABSTRACT

A transplant product made from human amniotic fluid has a supernatant from filtered and centrifuged amniotic fluid, the amniotic fluid when recovered aseptically having a clear, translucent to slightly pink or tan color. The supernatant had been taken from the amniotic fluid which had been passed through a 170 to 260 micron blood filter, then centrifuged for 5 minutes or more at 400 g and thereafter the supernatant was separated from a pellet of debris, leaving the biochemical properties intact, wherein the supernatant is cryofrozen in sized cryovials having 0.25 to 2.0 ml of the supernatant at a temperature of −65 degrees or less prior to use and the transplant products method of use. The transplant product contains cellular material, cell fragments, proteins and growth factors maintaining the biochemical properties and is non-immunogenic while having particles up to 170 microns being passable through a 30 gauge syringe for injection.

RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 16/503,747 filed on Jul. 5, 2019 entitled, “Liquid Amnion TransplantProduct”; which is a division of U.S. application Ser. No. 14/823,770now U.S. Pat. No. 10,413,572 issued on Sep. 17, 2019.

TECHNICAL FIELD

This invention relates to the manufacturing of a transplant productderived from human amniotic fluid (AF). The transplant product iscategorized as a liquid version of amniotic tissue.

BACKGROUND OF THE INVENTION

Within the uterus of a pregnant woman, a growing fetus is surrounded andcushioned by amniotic fluid, a watery liquid within the amnion Amnioticfluid is one of the main samples used for the medical examination of thepregnant woman and her fetus.

Traditionally, during a Caesarean section, after cutting through theuterus, the amniotic fluid will be suctioned away and discarded to makemore room Amniotic fluid contains cells, electrolytes, growth factors,carbohydrates, lipids, proteins, amino acids, lactate, pyruvate, enzymesand hormones Amniotic fluid is also a source of stem cells which ideallyshould be isolated and separately cultivated for cell therapy purposes.While amniotic fluid cells can be obtained from a small amount of fluidduring amniocentesis, these amounts are insufficient for a larger scaleharvesting of biomolecules or culturing of the stem cells comprisingamniotic fluid.

In US 2015/0025366 entitled “Method for Obtaining Sterile Human AmnioticFluid and Uses Thereof” published Jan. 22, 2015 stated, there is arecognized need in the art for an improved means for obtaining sterileamniotic fluid for use in research and the development of therapeuticproducts. Particularly, the prior art is deficient in methods forobtaining sterile human amniotic fluid with minimal or no risk to apregnant woman or fetus by collecting the amniotic fluid prior to anelective Caesarean section. Also the prior art is deficient in methodsfor obtaining sterile human amniotic fluid devoid of cells which maycreate unwanted reactions due to their allogenic characters on thepatients to be treated. The inventor, Harrell, claimed to fulfill thislongstanding need and desire in the art to improve the safety ofamniotic fluid in its medical uses and taught how to sterile filter thefluid.

The prior art method of obtaining sterile filtered human amniotic fluidfrom an individual. This method comprises the steps of obtaining sterilehuman amniotic fluid from an individual, removing cells, large particlesand other undissolvables from said human amniotic fluid by high speedcentrifugation, followed by membrane filtration. The first step is tocentrifuge the amniotic fluid in swing out buckets adapted to swing outrotors or other centrifugation bottles in angle rotors at about 5,000rpm to about 10,000 rpm for about 30 minutes to about 60 minutes. Thesupernatant is then filtered using filters with a pore size of about 5μm to about 10 μm to obtain the first filtrate, then filtering saidfirst filtrate through filters with a pore size of about 1.0 μm toobtain a second filtrate, filtering the second filtrate through filterswith the pore size of 0.45 μm or/and 0.2 μm to obtain a sterilelyfiltered amniotic fluid. In case of a final membrane filtration limitedto 0.45 μm, it is preferable to repeat a second filtration on a second0.45 μm membrane, to increase the sterility assurance level. The sterileamniotic fluid retains the growth factors from the raw amniotic fluid.In this method, the first centrifugation step may be replaced by depthfiltration through available filtration systems, however this option isnot preferred because it leads to important volume losses andundesirable adsorption of growth factors by the filtration media.

This technique disclosed by Harrell achieved a sterile fluid thatremoved all particles down to 0.2 micron. All cells and particulategreater than the 0.2 micron are removed, leaving a fluid devoid of muchof the beneficial biochemical particles needed to be useful in medicaltreatments.

The present invention discloses a method to recover amniotic fluid andmaintain particles in sizes up to 170 microns to 260 microns, over 100times greater, but maintaining a non-immunogenic product having superiorbiochemical properties suitable for direct injection into patients.

SUMMARY OF THE INVENTION

A method for a transplant product made from human amniotic fluid has thesteps of aseptically recovering a volume of greater than 100 ml of humanamniotic fluid from a woman, visualizing the fluid to confirm theremoved volume has a pink to clear color and contains no vernix prior tofiltering and then filtering said amniotic fluid through a blood filterwith a pore size of 170 to 260 microns. The process of filtering theamniotic fluid is performed to ensure the biochemical properties of thetissue remain intact. Placing the filtered amniotic fluid in a pluralityof centrifuge tubes by the step of transferring the filtered amnioticfluid tissues in the centrifuge tubes into a calibrated centrifuge;centrifuging at a centrifuge cycle of 5 minutes at 400 g. When the cycleis complete, the centrifuged fluid is separated into a supernatant fromthe amniotic fluid and a pellet containing debris; the supernatant iscarefully removed and separated from the pellet and set aside forfurther processing.

Thereafter, aliquoting the supernatant of liquid amnion into sizedcryovials of transplant product sizes in the range of 0.25 ml to 2.0 ml.A smaller volume of the 0.25 mL and 0.5 mL transplant product size isplaced into 1.8 mL cryovials and a larger volume 1.0 mL and 2.0 mLtransplant product size is placed into the larger 4.5 mL cryovials. Thevolumes are aliquoted into the appropriate sized cryovials using acalibrated pipette. Randomly selected filled cryovials are used forliquid microbiology cultures that represent the transplant product. Thecryovials after being filled with supernatant from the amniotic fluidare packaged, inspected and stored in the absence of a cryopreservativein a −65° C. or colder freezer. The sized volume of frozen supernatantwhen thawed and resuspended in a diluent, such as sterile water, for usehas a viscosity approximating water and is injectable through a 30 gaugeneedle. The supernatant of amniotic fluid when thawed is non-immunogenicand has preserved the biochemical propertied.

A method of treatment using the transplant product involves the steps oftaking a sterile syringe with at least a 30 gauge needle and filling itwith the thawed and resuspended supernatant of amniotic fluid andinjecting the fluid into the patient to be treated. The patient to betreated has one or more of the following conditions of damaged orinjured tissue, or a degenerative tissue condition and the injection ofthe supernatant from amniotic fluid is a treatment for said condition.

A transplant product made from human amniotic fluid has a supernatantfrom filtered and centrifuged amniotic fluid, the amniotic fluid whenrecovered aseptically having a clear, translucent to slightly pink ortan color. The supernatant had been taken from the amniotic fluid whichhad been passed through a 170 to 260 micron blood filter, thencentrifuged for 5 minutes or more at 400 g and thereafter thesupernatant was separated from a pellet of debris, leaving thebiochemical properties intact, wherein the supernatant is cryofrozen insized cryovials having 0.25 to 2.0 ml of the supernatant at atemperature of −65 degrees or less prior to use. The transplant productcontains cell or cell fragments, proteins and growth factors maintainingthe biochemical properties and is non-immunogenic while having particlesup to 170 microns being passable through a 30 gauge syringe forinjection.

Definitions

Vernix caseosa, also known as vernix, is the waxy or cheese-like whitesubstance found coating the skin of newborn human babies. Vernix startsdeveloping on the baby in the womb around 18 weeks into pregnancy.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by way of example and with reference tothe accompanying drawings in which:

FIG. 1 is a photograph of amniotic fluid that is being asepticallytransferred to a blood bag.

FIG. 2 is a photograph showing the amniotic fluid in the blood bag beingfiltered into centrifuge tubes.

FIG. 3 is a photograph showing centrifuge tubes after being filled withthe filtered amniotic fluid before being centrifuged.

FIG. 4 is a photograph showing the liquid supernatant from the amnioticfluid after the centrifuge process.

FIG. 5 is a photograph showing frozen supernatant from the amnioticfluid after the centrifuge process.

FIG. 6 is a photograph showing a clear supernatant from the amnioticfluid after the centrifuge process.

FIG. 7 is a flow chart detailing the filtering process of the amnioticfluid.

FIG. 8 is a flow chart detailing the centrifugation process of theamniotic fluid.

FIG. 9 is a flow chart detailing the aliquoting process of the amnioticfluid.

FIG. 10 is a perspective view of a syringe with the transplant product.

DETAILED DESCRIPTION OF THE INVENTION

With reference to photographs 1-3, technicians are shown with a bloodbag 10 filled with a volume of amniotic fluid 2 which will be processedthrough a blood filter 16 and collected into small containers 20. Asshown in FIG. 1, the technician is pipetting a volume of the amnioticfluid 2 into a syringe connected by tubing which is thereby filling theblood bag 10. The blood bag 10 is then connected to an IV tube or set 14with a blood filter 16 at the bottom of the drip chamber and then thefluid 2 is delivered to a centrifuge container 20, as shown in FIG. 2.As shown in FIG. 3, each technician is holding a container of theamniotic fluid. As shown in the photographs, the amniotic fluid 2recovered from a pregnant woman who had elected cesarean is shown with aslightly reddish color, this color in the photograph is unacceptable. Inpractice, the amniotic fluid 2 should be clear, translucent, slightlypink or slightly tan. The deep red color as shown is exemplary of anunacceptable volume of amniotic fluid 2 because it has too much bloodproduct and debris. Properly collected amniotic fluid 2 will besubstantially clearer than that as illustrated in FIGS. 1-3. For thepurposes of this invention, when the amniotic fluid 2 is recoveredproperly in an aseptic technique, and used during the filtering stepexplained in diagram 7 prior to centrifuging, the material will have thecolor as indicated which should be confirmed to be clear, translucent,slightly pink or slightly tan.

With reference to FIG. 4, the material once centrifuged, is separatedwherein a supernatant 4 is formed along with a solid pellet of debris.The supernatant 4 is carefully pipetted out from the container 20 aftercentrifuging leaving the debris. An important aspect of the filteringshown in the photographs of FIGS. 1-3 is that the porosity of the bloodfilter 16 used is approximately 170 to 260 microns. This means thatparticles and cell fragments below 170 microns are clearly passedthrough the filter 16 to be centrifuged. Some as high as 260 microns canpass through the filter 16 depending on the filter pore size. In thisrange, it has been found that a large volume of the particularlybeneficial cell fragments and cellular material can be passed into thefiltered amniotic fluid. At this point the amniotic fluid 2 is movedinto centrifuge tubes 20. The centrifuge tubes 20 receive a volume ofmaterial to be spun for a short duration at a relatively low ormoderately low speed. Once the material is effectively spun for 5minutes at 400 g, the pellet is formed in the bottom of the tube and thesupernatant 4 can be pipetted off as discussed. Once this occurs, thesupernatant 4 is carefully removed and the centrifugation process isfinished as diagrammed in FIG. 8. Once the aliquoting process iscompleted and the supernatant 4 is divided into cryovials 40, it isrecommended that 0.25 to 0.5 ml supernatant 4 material be placed insmall vials 40 as shown in FIGS. 4-6. The small vials 40 areapproximately 1.8 ml in volume. Larger volumes from 0.1 to 2.0 ml can bemaintained in larger cryovials 40 of 4 ml or greater. These are thencryogenically frozen. FIG. 4 shows the supernatant 4 directly aftercentrifuge process wherein FIG. 5 shows the frozen supernatant 4 fromthe amniotic fluid 2 after the centrifuge process. FIG. 6 shows a clearsupernatant 4 from the amniotic fluid 2 after the centrifuge process.

As shown, the frozen or liquid supernatant 4 taken from the filtered andcentrifuged amniotic fluid 2 can have a very light pink to translucentcolor either in the frozen or the liquid form. This indicates that thematerial contains no immunological artifacts that could cause a problemwhen injected into a patient. As illustrated, the finished transplantproduct 100 will be non-immunogenic and maintain the biochemicalproperties found in the naturally occurring amniotic fluid. Theadvantage of the present invention is that a much larger array ofparticles and cell fragments and cellular material from the amnioticfluid will be passed through the blood filter and yet surprisingly, dueto the processing technique this material will maintain its sterilityand its non-immunogenic characteristics if properly handled usingaseptic techniques described in the present invention. If however, thematerial is allowed to be collected and recovered wherein the fluidcolor is reddish or dark reddish, it is noted that this material wouldnot be a proper source of amniotic fluid from which a transplantableproduct 100 can be manufactured.

The benefits of the above referenced transplant product 100 made fromthe supernatant of the filtered and centrifuged properly colored orclear amniotic fluid is that it can be used to treat a variety ofconditions, in particular skin conditions or any soft tissue conditionswherein a regenerative outcome is anticipated or desired. In the case ofdegenerative material, it is possible that injecting the amniotic fluidin the region to be treated will have a beneficial outcome with regardto the condition. Under these circumstances, it has been determined thatthe transplantable material ideally should be suitable for injectiondirectly into a patient when resuspended in a diluent after thawing. Tohave this occur, the filtration was set such that the particle sizewould not occlude or block a small gauge needle such as a 30 gaugeneedle 32. Accordingly, in preparing the supernatant 4, from its thawedunfrozen stated it is recommended that it be diluted with sterile wateror other suitable diluent to form the liquid amnion transplant product100 which is then drawn through a syringe 30 as shown in FIG. 10, forlater injection into the patient. Preferably, this is done on site andat the time of the injection. Once this is accomplished, the transplantproduct 100 collected in the syringe 30 can be injected directly intothe patient into the area to be treated or can be provided as a topicalspray through a spray nozzle attached to the syringe or other dispensingdevice.

The advantage of the present invention as compared to the prior art isthat the centrifugation only occurs in 5 minutes is accomplished at alow rpm of 400 g and this removes the unwanted and undesirable debrisparticles and blood cells from the amniotic fluid. As a result, thematerial is left in a rather pristine state, unaltered biochemically bythe processing techniques. The prior art recommended filtration down to0.2 microns which will eliminate any microbiological remnants from thefluid, however, in doing so a tremendously large volume of thebeneficial biochemical properties of the amniotic fluid is screened andfiltered from the resultant supernatant. Furthermore, the spinning ofthe supernatant at 10 times the gravity recommended in the presentinvention, means that a larger debris button is created. The end productis an amniotic fluid that basically approaches the value of sterilewater. This leaves very little of the constituent biologic value for theproduct. In the prior art mentioned in the background of this invention,it is noted that the supernatant made from the amniotic fluid was usedas an eye droplet and therefore very minor improvements in thebiochemical properties compared to water may be beneficial for thetreatment of an eye as the larger particles in the present product 100could be adverse to surface treating an eye. In most other areas where amore meaningful and more aggressive treatment is expected, it isbelieved that the present invention provides orders of magnitudesuperior biochemical properties including growth factors that will helpin the regeneration of tissue.

Developmental research testing has confirmed the existence of preservedgrowth factors including TIMP-2, HGF, TIMP-3, TIMP-1, IGF-2, IL-6,IGF-1, GRO-alpha, TGF-B2, TGF-B1, IL-1RA, TIMP-4, MCP-1, EGF, TGF-alphaand TNF-alpha that aid in wound healing.

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed, which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. A liquid amnion transplant product comprising asupernatant from filtered and centrifuged amniotic fluid: wherein theamniotic fluid when recovered aseptically has a clear, translucent toslightly pink or tan color; wherein the supernatant having been takenfrom the amniotic fluid which had passed through a 170-260 micron bloodfilter; wherein the filtered amniotic fluid then having been centrifugedand the supernatant is further separated from a pellet of debrisparticles of a size as high as 260 microns, leaving the supernatantcontaining cellular material or cell fragments set at a particle size sothe isolated supernatant does not block a 30 gauge needle (152 microns)and retains biochemical properties.
 2. The liquid amnion transplantproduct of claim 1, wherein the product is cryofrozen in a volume of0.25 to 2.0 ml in a cryovial.
 3. A method for producing a transplantproduct, comprising the steps of: a) obtaining a volume of greater than100 ml of sterile human amniotic fluid from a woman, wherein the volumehas a pink to clear color and contains no vernix; b) filtering theamniotic fluid through a blood filter with a pore size of 170 to 260microns; c) and centrifuging the filtered amniotic fluid to produce apellet of debris particles of a size as high as 260 microns and asupernatant that is the transplant product, wherein the supernatantcontains cellular material or cell fragments of a particle size lessthan 260 microns, wherein the supernatant does not block a 30 gaugeneedle (152 microns), and wherein the supernatant has the biochemicalproperties of the tissue intact.